Angiogenesis is a fundamental process by which new blood vessels are formed, as reviewed, for example, by Folkman and Shing, J. Biol. Chem. 267 (16), 10931-10934 (1992). It is essential in reproduction, development and wound repair. Under these conditions, angiogenesis is highly regulated, so that it is turned on only as necessary, usually for brief periods of days, then completely inhibited. However, many diseases are driven by persistent unregulated angiogenesis. In arthritis, new capillary blood vessels invade the joint and destroy cartilage. In diabetes, new capillaries invade the vitreous, bleed, and cause blindness. Ocular neovascularization is the most common cause of blindness. Tumor growth and metastasis are angiogenesis-dependent. A tumor must continuously stimulate the growth of new capillary blood vessels for the tumor itself to grow.
Capillary blood vessels consist of endothelial cells and pericytes. These two cell types carry all of the genetic information to form tubes, branches and whole capillary networks. Specific angiogenic molecules can initiate this process. Specific inhibitory molecules can stop it. These molecules with opposing function appear to be continuously acting in concert to maintain a stable microvasculature in which endothelial cell turnover is thousands of days. However, the same endothelial cells can undergo rapid proliferation, i.e. less than five days, during burst of angiogenesis, for example, during wound healing.
A number of proteases have been implicated as key factors in angiogenesis. See, for example, Mignatti, et al., Cell 47, 487-498 (1986) and Rifkin, et al., Acta. Biol. Med. Germ. 40, 1259-1263 (1981), who suggest several enzymes in a proteolytic cascade, including plasminogen activator and collagenase, must be inhibited in order to inhibit angiogenesis.
Under normal conditions, angiogenesis is associated with such events as wound healing, corpus luteum formation and embryonic development, as discussed by Folkman, et al., Science 43, 1490-1493 (1989). However, a number of serious diseases are also dominated by abnormal neovascularization including solid tumor growth and metastases, some types of eye disorders, and rheumatoid arthritis, reviewed by Auerbach, et al., J. Microvasc. Res. 29, 401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, pp. 175-203 (Academic Press, New York 1985); Patz, Am. J. Opthalmol. 94, 715-743 (1982); and Folkman, et al., Science 221, 719-725 (1983). For example, there are a number of eye diseases, many of which lead to blindness, in which ocular neovascularization occurs in response to the diseased state. These ocular disorders include diabetic retinopathy, neovascular glaucoma, inflammatory diseases and ocular tumors (e.g. retinoblastoma). There are a number of other eye diseases which are also associated with neovascularization, including retrolental fibroplasia, uveitis, approximately twenty eye diseases associated with choroidal neovascularization and approximately forty eye diseases which are associated with iris neovascularization. The current treatment of these diseases is inadequate, especially once neovascularization has occurred, and blindness often results.
Key components of the angiogenic process are the degradation of the basement membrane, the migration and proliferation of capillary endothelial cell (EC) and the formation of three dimensional capillary tubes. The normal vascular turnover is rather low: the doubling time for capillary endothelium is from 50-20,000 days, but it is 2-13 days for tumor capillary endothelium. The current understanding of the sequence of events leading to angiogenesis is that a cytokine capable of stimulating endothelial cell proliferation, such as fibroblast growth factor (FGF), causes release of collagenase or plasminogen activator which, in turn, degrade the basement membrane of the parent venule to facilitate in the migration of the endothelial cells. These capillary cells, having `sprouted` from the parent vessel, proliferate in response to growth factors and angiogenic agents in the surrounding to form lumen and eventually new blood vessels. Thus, inhibition of angiogenesis can occur at any of the above key junctures. A chemical agent which prevents the continued spread of vascularization could have broad applicability as a therapy for those disease in which neovascularization plays a prominent role.
Heparin and heparan sulfate represent a class of glycosaminoglycans characterized by a linear polysaccharide of D-glucosamine (1.fwdarw.) linked to hexuronic acid (Linhardt, R. J. (1991) Chem. Ind. 2, 45-50; Casu, B. (1985) Adv. Carbohydr. Chem. Biochem. 43, 51-134). Heparin and heparan sulfate are complex carbohydrates that play an important functional role in the extracellular matrix of mammals. These polysaccharides modulate and regulate tissue level events that take place either during development under normal situations or wound healing and tumor metastasis under pathological conditions.
Much of the current understanding of heparin and heparan sulfate sequence has relied on studies of their biosynthesis (Linhardt, R. J., Wang, H. M., Loganathan, D., and Bae, J. H. (1992) Biol. Chem. 267, 2380-2387; Lindahl, U., Feingold, D., and Roden, L. (1986) Trends Biochem. Sci. 11, 221-225; Jacobson, I., and Lindahl U. (1980) J. Biol. Chem. 255, 5094-5100; Lindahl, U., and Kjellen, L. (1987) in The Biology of Extracellular Matrix Proteoglycans (Wight, T. N., and Mecham R., eds) pp. 59-104, Academic Press, New York).
Heparan sulfate, which is chemically almost indistinguishable from heparin, is believed to be present on virtually all cell surfaces. Heparin-like molecules are associated with membrane proteins and are called proteoglycans. Proteoglycans are predominantly found in the extracellular matrix (ECM) and function in cell adhesion to the extracellular matrix. It is increasingly recognized that heparin is more than a mere structural oligosaccharide as it interacts with other key proteins of the extracellular matrix, such as laminin, fibronectin and collagen, and helps to define the physiological properties of the matrix. Heparin interacts with an array of cytokine-like growth factors present in the extracellular matrix, by facilitating their biochemical interaction with receptors and by protecting them from proteolytic degradation. Heparin potentiates the biological activity of aFGF, as reported by Thornton, et al., Science 222, 623-625 (1983), possibly by potentiating the affinity of aFGF for its cell surface receptors, as reported by Schreiber, et al., Proc. Natl. Acad. Sci. USA 82, 6138-6142 (1985). Heparin protects aFGF and bFGF from degradation by heat, acid and proteases, as reported by Gospodarowicz and Cheng, J. Cell Physiol. 128, 475-4 84 (1986); Rosengart, et al., Biochem. Biophys. Res. Commun. 152, 432-440 (1988); and Lobb Biochem. 27, 2572-2578 (1988). bFGF is stored in the extracellular matrix and can be mobilized in a biologically active form by heparin or heparan sulfate, as reported by Vlodavsky, et al., Proc. Natl. Acad. Sci. USA 84, 2292-2296 (1987) and Folkman, et al., Am. J. Pathol. 130, 393-400 (1988). The binding of FGF to heparan sulfate is a prerequisite for the binding of FGF to its high affinity receptor on the cell surface, as reported by Yayon, et al., Cell 64, 841-848 (1991) and Papraeger, et al., Science 252, 1705-1708 (1991). A specific heparan sulfate proteoglycan has been found to mediate the binding of bFGF to the cell surface, as described by Kiefer, et al., Proc. Natl. Acad. Sci. USA 87, 6985-6989 (1990).
Although a number of these studies have focused on the role of heparin-like molecules in neovascularization, little is known about the role of heparin-degrading enzymes in neovascularization. Heparin-like molecules such as heparin and heparan sulfate bind several cytokines, which are angiogenic, and modulate their function either by stabilizing them or by controlling their bioavailability, as reported by Folkman and Shing, J. Biol. Chem. 267, 10931-10934 (1992). These molecules have been shown by Klagsbrun and Baird Cell 67, 229-231 (1991), to act as low affinity receptors on cell surfaces and to facilitate growth factor activity and receptor binding.
These observations suggest that enzymes which degrade heparin-like molecules can play a role in modulating neovascularization. Far less is known about the direct role of heparinase on the angiogenic process than is known about that of its substrate, heparin.
Heparin lyases are a general class of enzymes that are capable of specifically cleaving the major glycosidic linkages in heparin and heparan sulfate. Three heparin lyases have been identified in Flavobacterium heparinum, a heparin-utilizing organism that also produces exoglycouronidases, sulfoesterases, and sulfamidases that further act on the lyase-generated oligosaccharide products (Yang, et al. J. Biol. Chem. 260, 1849-1857 (1987); Galliher, et al. Eur. J. Appl. Microbiol. Biotechnol. 15, 252-257 (1982). These lyases are designated as heparin lyase I (heparinase, EC 4.2.2.7), heparin lyase II (heparinase II, no EC number) and heparin lyase III (heparitinase EC 4.2.2.8). The three purified heparin lyases differ in their capacity to cleave heparin and heparan sulfate: Heparin lyase I primarily cleaves heparin, heparin lyase III specifically cleaves heparan sulfate and heparin lyase II acts equally on both heparin and heparan. Several Bacteroides sp. (Saylers, et al. Appl. Environ. Microbiol. 33, 319-322 (1977); Nakamura, et al. J. Clin. Microbiol. 26, 1070-1071 (1988)) also produce heparinases. A heparinase has also been purified to apparent homogeneity from an unidentified soil bacterium by Bohmer, et al.J. Biol. Chem. 265, 13609-13617 (1990).
The in vivo effect of these heparinases, other than on the degradation of heparin, has never been determined.
It is an object of the present invention to provide pharmaceutical compositions, and method of use thereof, based on heparinases, for the treatment of diseases involving abnormal angiogenesis.
It is a further object of the present invention to provide pharmaceutical compositions, and method of use thereof, based on heparinases, for inhibition of capillary endothelial cell proliferation and migration.
It is another object of the present invention to provide topical and controlled release pharmaceutical compositions, and methods of use thereof, based on heparinases, for inhibition of angiogenesis.